Factor VII-Induced MicroRNA-135a Inhibits Autophagy and Is Associated with Poor Prognosis in Hepatocellular Carcinoma  Kuang-Tzu Huang, I-Ying Kuo, Ming-Chao Tsai, Chun-Hsien Wu, Li-Wen Hsu, Li-Yu Chen, Chao-Pin Kung, Yu-Fan Cheng, Shigeru Goto, Yu-Wei Chou, Chao-Long Chen, Chih-Che Lin, Kuang-Den Chen  Molecular Therapy - Nucleic Acids  Volume 9, Pages 274-283 (December 2017) DOI: 10.1016/j.omtn.2017.10.002 Copyright © 2017 The Authors Terms and Conditions

Figure 1 miR-135a Is Upregulated in HCC Tumor with High FVII Expression (A) Expression of miR-135a was compared between HCC tumors (T) and their contiguous normal regions (N). (B) HCC cell line Hep3B was treated with recombinant TF, FVIIa, or a PAR2 peptide agonist for 24 hr. The level of miR-135a was analyzed. (C) Hep3B cells were transfected with siRNAs for TF, FVII, or PAR2. Expression of miR-135a was examined after 24 hr. (D) Hep3B cells were transfected with mTOR siRNA. The level of miR-135a was evaluated after 24 hr. (E) (Left) Correlations between FVII and miR-135a levels in HCC and non-tumor counterparts were assessed. We grouped patients having higher FVII and miR-135a levels in the tumor than the normal region (+/+) with patients having lower FVII and decreased miR-135a (−/−). These cases were compared with those having negatively correlated levels of FVII and miR-135a (+/− and −/+). (Right) Tumor/normal ratio of miR-135a expression was compared using qRT-PCR in HCC tumors that had higher FVII levels than the adjacent normal region (FVII T > N) and those that had lower FVII than the normal region (FVII T < N). The data are presented as mean ± SD. Statistically significant compared with controls at *p < 0.05; **p < 0.01; ***p < 0.001. Molecular Therapy - Nucleic Acids 2017 9, 274-283DOI: (10.1016/j.omtn.2017.10.002) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Atg14 Is a Direct Target of miR-135a (A) Schematic representation of predicted miR-135a binding sequence at the 3′ UTR of atg14 gene. (B) Hep3B cells were transfected with FVII siRNA for 24 hr. FVII protein (left) and Atg14 mRNA (right) levels were determined. (C) HCC cell lines Hep3B (left) and HepG2 (right) were transfected with a miR-135a mimic (20 nM, 40 nM), and the mRNA level of Atg14 was evaluated after 24 hr. (D) Hep3B cells were co-transfected with a miR-135a mimic and a reporter construct that contained the wild-type (WT) or mutated (MT) miR-135a binding sequence. Post-transcriptional repression was determined by measuring the relative luciferase activity. (E) Correlations of HCC tumor/normal ratios of Atg14 and miR-135a expression were depicted. (F) Kaplan-Meier probability distributions showing disease-free survival according to miR-135a levels in HCC tumors compared with their paired non-tumor tissues. The data are presented as mean ± SD. Statistically significant compared with controls at *p < 0.05; ***p < 0.001. Molecular Therapy - Nucleic Acids 2017 9, 274-283DOI: (10.1016/j.omtn.2017.10.002) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Combining Relative miR-135a Expression with AFP Levels Better Predicts Tumor Recurrence Than Assessing Using AFP Alone Of the 103 HCC cases, patients were divided according to their preoperative AFP levels (cut-off = 70 ng/mL). Each group was then subcategorized based on the relative miR-135a expression in tumors compared to the adjacent normal areas (T > N or T < N). The number at the bottom of each box indicates case counts. R, recurrence; NR, non-recurrence. Molecular Therapy - Nucleic Acids 2017 9, 274-283DOI: (10.1016/j.omtn.2017.10.002) Copyright © 2017 The Authors Terms and Conditions

Figure 4 miR-135a Inhibits the Autophagy Pathway In Vitro (A) Hep3B and HepG2 cells were transfected with a miR-135a mimic (final concentrations, 10 nM, 20 nM), and protein levels of Atg14 and LC3A/B were determined after 24 hr using immunoblotting. (B) Protein levels of Atg5, Atg7, and Atg12 were examined in miR-135a-transfected Hep3B cells. (C) Hep3B cells were transfected with a miR-135a mimic (final concentrations, 20 nM, 40 nM) for 24 hr. Protein levels of LAMP-1, cathepsin D, and LC3A/B were determined. Precursor and mature forms of cathepsin D are indicated. Densitometric values are presented as fold changes over the controls (set as 1) and are shown below each lane of the blot. Molecular Therapy - Nucleic Acids 2017 9, 274-283DOI: (10.1016/j.omtn.2017.10.002) Copyright © 2017 The Authors Terms and Conditions